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Abstract

Lipopolysaccharide is the major constituent of the outer membrane of gram-negative bacteria and, once released from the bacterial surface into the bloodstream, is a potent activator of the host immune system, which can lead to septic shock. LPS has a hydrophilic region consisting of a repeating oligosaccharide that is strain-specific (O-antigen) and a core polysaccharide, which is covalently linked to a hydrophobic lipid moiety (lipid A). Lipid A is the most conserved part and is responsible for the toxicity of LPS. Therefore, finding molecules able to bind to this region and neutralize LPS toxicity is of relevant interest as it may provide new therapies to prevent septic shock (Chen et al., 2006). Several proteins and peptides were reported to bind LPS and alter its toxicity towards reduction and even enhancement (Brandenburg et al., 1998), such as serum albumin (Ohno and Morrison, 1989), lipopolysaccharide binding protein (LBP) (de Haas et al., 1999), casein (López-Expósito et al., 2008), lysozyme, the antibiotic polymyxin B and antimicrobial peptides (Chen et al., 2006). Although some of these proteins are neutral and even anionic/acidic (pI<7) (Jang et al., 2009), due to the amphipathic structure of LPS and the presence of negatively charged phosphate groups on the lipid A, the most important factors that are considered for optimal binding to LPS are a cationic/basic (pI>7) and amphipathic nature (Chen et al., 2006). Here we describe a competitive ELISA that can be used to identify proteins or peptides that bind LPS, as a first approach before analyzing the possible activity in vitro and in vivo. In this ELISA, serial dilutions of the protein or peptide to be tested are preincubated with a fixed concentration of fluorescein isothiocyanate (FITC)-labeled LPS from Escherichia coli serotype O111:B4 and then added to wells of a microtitre plate which are blocked with a casein hydrolysate that binds LPS (Martínez-Sernández et al., 2014). Binding of the protein to LPS displaces LPS from binding to the casein, which is revealed using a horseradish peroxidase (HRP)-labeled anti-FITC polyclonal conjugate. This method allows simultaneous analysis of several proteins or peptides in a short period of time and no recognizing molecules (e.g., antibodies) to a specific protein or peptide are needed.

Determine the number of wells needed for the assay. The test should be performed at least in duplicate. Following the ELISA worksheet example below, if you test in duplicate 8 serial dilutions of an unknown protein/peptide plus the same dilutions of polymyxin B (positive inhibition control) and a known protein/peptide with no binding ability to LPS (negative inhibition control) you need 52 wells: 3 x 8 x 2 = 48 wells for protein/peptide dilutions, two wells with LPS-FITC (reference control) and two wells without LPS-FITC (negative control).

Add 200 µl of casein hydrolysate solution to the wells of a 96-well microplate. Seal the microplate with the adhesive film and incubate overnight at 4 °C without shaking.

Separately, prepare a 2x solution of LPS O111: B4-FITC conjugate (5 µg/ml) and 2x serial dilutions of the protein/peptide to be tested (starting dilution 80 µg/ml) in PBS-EDTA. Prepare the same dilutions of polymyxin B (positive inhibition control) and a negative inhibition control (e.g., ovalbumin). Eight serial ¼ dilutions are optimal for most proteins. For the ELISA worksheet example prepare:

2x solution of LPS-FITC: 3.5 ml of LPS-FITC at 5 µg/ml in PBS-EDTA.

2x serial dilutions of the proteins/peptides in PBS-EDTA:

Table 1. Preparation of serial dilutions

Dilution (dil) number

Volume and source of proteins/peptides

Volume of PBS-EDTA

Protein concentration

1

3.2 μl from a 5 mg/ml stock

196.8 μl

80 µg/ml

2

50 μl of dil 1

150 μl

20 µg/ml

3

50 μl of dil 2

150 μl

5 µg/ml

4

50 μl of dil 3

150 μl

1.25 µg/ml

5

50 μl of dil 4

150 μl

0.312 µg/ml

6

50 μl of dil 5

150 μl

0.078 µg/ml

7

50 μl of dil 6

150 μl

0.020 µg/ml

8

50 μl of dil 7

150 μl

0.005 µg/ml

Mix 120 µl of LPS-FITC solution (2x) with 120 µl of each dilution (2x) or PBS-EDTA (reference control) in individual microcentrifuge tubes, mix well, and incubate the samples for 1 h at RT.

Aspirate the content of the wells coated with the casein hydrolysate and wash 3 times with 200 µl of PBS (no soak) at RT without shaking.

Immediately, add 100 µl of the preincubated solutions or PBS-EDTA (negative control) to each well in duplicate. Seal the microplate and incubate for 30 min at RT under shaking at 750 rpm.

Wash the plate 5 times with 200 µl of PBS-T (no soak) at RT without shaking.

Add 100 µl of sheep anti-FITC: HRP diluted 1/4,000 in PBS-T. Seal the microplate and incubate for 30 min at RT under shaking at 750 rpm.

Add 100 µl of the OPD solution to each well and incubate for 20 min at RT, without shaking and in the dark.

Add 25 µl of 3 N H2SO4 to each well to stop the reaction.

Read the absorbance of the wells at 492 nm within 30 min.

The percentage of inhibition for each protein dilution is calculated according to the formula: [(average OD reference control) - (average OD protein dil + LPS-FITC)]/ (average OD reference control) x 100. OD: Optical density.
*Alternatively, incubations in steps 6 and 8 can be performed in an incubator for 1 h at 37 °C without shaking, after sealing the plate with the adhesive film.

Table 2. ELISA worksheet example

1

2

3

4

5

6

7

A

100 µl LPS-FITC (reference control)

100 µl

Protein dil 1 + LPS-FITC

100 µl

PolymyxinB dil 1 + LPS-FITC

100 µl

Ovalbumin dil 1 + LPS-FITC

B

100 µl

Protein dil 2 + LPS-FITC

100 µl

Polymyxin B dil 2 + LPS-FITC

100 µl

Ovalbumin dil 2 + LPS-FITC

C

100 µl

PBS-EDTA

(negative control)

100 µl

Protein dil 3 + LPS-FITC

100 µl

Polymyxin B dil 3 + LPS-FITC

100 µl

Ovalbumin dil 3 + LPS-FITC

D

100 µl

Protein dil 4 + LPS-FITC

100 µl

Pol ymyxin B dil 4 + LPS-FITC

100 µl

Ovalbumin dil 4 + LPS-FITC

E

100 µl

Protein dil 5 + LPS-FITC

100 µl

Polymyx in B dil 5 + LPS-FITC

100 µl

Ovalbumin dil 5 + LPS-FITC

F

100 µl

Protein dil 6 + LPS-FITC

100 µl

Polymyxin B dil 6 + LPS-FITC

100 µl

Ovalbumin dil 6 + LPS-FITC

G

100 µl

Protein dil 7 + LPS-FITC

100 µl

Polymyxin B dil 7 + LPS-FITC

100 µl

Ovalbumin dil 7 + LPS-FITC

H

100 µl

Protein dil 8 + LPS-FITC

100 µl

Polymyxin B dil 8 + LPS-FITC

100 µl

Ovalbumin dil 8 + LPS-FITC

Representative data

Figure 1. Example of inhibition curve obtained with several molecules using the reported method to analyze LPS binding. LPS-FITC (0.25 µg/well) was incubated with four-fold dilutions of polymyxin B (circles), LBP human recombinant (squares), bovine serum albumin (BSA) (inverted triangles) and myoglobin from equine skeletal muscle (triangles). It is noteworthy that an excess of protein/peptide might have a “zone effect” in the assay, as occurs with polymyxin B.

Notes

The ideal OD range is 0.6-1.2 for the reference control, which was obtained using LPS-FITC at 0.25 µg/well. As the FITC content may vary between batches this concentration might need to be adjusted before performing the inhibition assay. The extent of labeling of the LPS-FITC used to develop this method was of 7.20 µg FITC/mg LPS.

Binding of a protein to LPS does not imply that it is linked to a biological effect. Further experiments should be done to test the activity in vitro and in vivo.

Depending on several factors (amino acid composition, type of interaction with LPS, number of binding sites, etc.) each protein or peptide needs a different optimal concentration to obtain the maximal inhibition. Hence, several dilutions of the proteins or peptides need to be assayed.

The main advantage of using a competitive ELISA instead of an indirect ELISA is that in the latter, the protein/peptide to be tested is coated to the wells and blocking agents may interfere with LPS binding (e.g., binding LPS per se, causing steric hindrance). Casein hydrolysate is commonly used as a blocking agent in ELISA and also serves as LPS binding agent, enabling its use in the competitive system reported.

Inhibition will occur if the protein or peptide to be tested binds to the same or a close LPS region as the casein hydrolysate. Although casein hydrolysate is composed of several derived peptides and therefore the mechanism of binding to LPS is poorly understood, as polymyxin B inhibits LPS binding to casein, this suggests that the casein hydrolysate binds at least to the lipid A region of LPS.

LPS is incubated in PBS-EDTA in the absence of detergents as both LPS and detergents have an amphipathic nature and may give unexpected results difficult to interpret when incubated together.

Recipes

Casein hydrolysate solutionNote: The preparation is based on the method described by Pearce-Pratt and Roser (2010) with some modifications.

Dissolve 2.5 g of casein in 80 ml of 0.3 M NaOH and incubate overnight at 37 °C under shaking at 150 rpm

Adjust pH to 8 slowly with 7.4% HCl Note: Aggregation of casein fragments occurs transiently while the pH
is adjusted. Keep the pH meter electrode out of the solution when you
add HCl. Read the pH or add more HCl dropwise always after the
aggregates are dissolved.

Add 2 ml of concentrated PB (the pH lowers to approximately 7.5)

Adjust pH to 7.2 with 7.4% HCl

Made up to 200 ml with distilled water and readjust the pH if necessary